Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

被引:14
作者
Goncalves, Manuel A. F. V. [1 ]
van Nierop, Gijsbert P. [1 ]
Holkers, Maarten [1 ]
de Vries, Antoine A. F. [1 ]
机构
[1] Leiden Univ, Med Ctr, Dept Mol Cell Biol, NL-2333 ZC Leiden, Netherlands
关键词
DOUBLE-STRAND BREAKS; SITE-SPECIFIC RECOMBINATION; INTERFERON-INDUCIBLE GENE; ADENOASSOCIATED VIRUS; MUSCLE-CELLS; INTEGRATION; GENOME; REPAIR; TRANSCRIPTION; SPECIFICITY;
D O I
10.1093/nar/gkr1234
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The exchange of genetic information between donor and acceptor DNA molecules by homologous recombination (HR) depends on the cleavage of phosphodiester bonds. Although double-stranded and single-stranded DNA breaks (SSBs) have both been invoked as triggers of HR, until very recently the focus has been primarily on the former type of DNA lesions mainly due to the paucity of SSB-based recombination models. Here, to investigate the role of nicked DNA molecules as HR-initiating substrates in human somatic cells, we devised a homology-directed gene targeting system based on exogenous donor and chromosomal target DNA containing recognition sequences for the adeno-associated virus sequence- and strand-specific endonucleases Rep78 and Rep68. We found that HR is greatly fostered if a SSB is not only introduced in the chromosomal acceptor but also in the donor DNA template. Our data are consistent with HR models postulating the occurrence of SSBs or single-stranded gaps in both donor and acceptor molecules during the genetic exchange process. These findings can guide the development of improved HR-based genome editing strategies in which sequence- and strand-specific endonucleolytic cleavage of the chromosomal target site is combined with that of the targeting vector.
引用
收藏
页码:3443 / 3455
页数:13
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